Many blood-ingesting pests are known to feed on humans and animals, and many pests are vectors for pathogenic microorganisms which threaten human and animal health, including commercially important livestock, pets and other animals. Various species of mosquitoes, for example, transmit diseases caused by viruses, and many are vectors for disease-causing nematodes and protozoa. Mosquitoes of the genus Anopheles transmit Plasmodium, the protozoan which causes malaria, a devastating disease which results in approximately 1 million deaths annually. The mosquito species Aedes aegypti transmits an arbovirus that causes yellow fever in humans. Other arboviruses transmitted by Aedes species include the causative agents of dengue fever, eastern and western encephalitis, Venezuelan equine encephalitis, St. Louis encephalitis, chikungunya, oroponehe and bunyarnidera. The genus Culex, which includes the common house mosquito C. pipiens, is implicated in the transmission of various forms of encephalitis and filarial worms. The common house mosquito also transmits Wuchereria banuffi and Brugia malayi, which cause various forms of lymphatic filariasis, including elephantiasis. Trypanasomas cruzi, the causative agent of Chagas"" disease, is transmitted by various species of blood-ingesting Triatominae bugs. The tsetse fly (Glossina spp.) transmits African trypanosomal diseases of humans and cattle. Many other diseases are transmitted by various blood-ingesting pest species. The order Diptera contains a large number of blood-ingesting and disease-bearing insects, including, for example, mosquitoes, black flies, no-see-ums (punkies), horse flies, deer flies and tsetse flies.
Various pesticides have been employed in efforts to control or eradicate populations of disease-bearing pests, such as disease-bearing blood-ingesting. pests. For example, DDT, a chlorinated hydrocarbon, has been used in attempts to eradicate malaria-bearing mosquitoes throughout the world. Other examples of chlorinated hydrocarbons are BHC, lindane, chlorobenzilate, methoxychlor, and the cyclodienes (e.g., aldrin, dieldrin, chlordane, heptachlor, and endrin). The long-term stability of many of these pesticides and their tendency to bioaccumulate render them particularly dangerous to many non-pest organisms.
Another common class of pesticides is the organophosphates, which is perhaps the largest and most versatile class of pesticides. Organophosphates include, for example, parathion, Malathion(trademark), diazinon, naled, methyil parathion, and dichlorvos. Organophosphates are generally much more toxic than the chlorinated hydrocarbons. Their pesticidal effect results from their ability to inhibit the enzyme cholinesterase, an essential enzyme in the functioning of the insect nervous system. However, they also have toxic effects on many animals, including humans.
The carbamates, a relatively new group of pesticides, include such compounds as carbamyl, methomyl, and carbofuran. These compounds are rapidly detoxified and eliminated from animal tissues. Their toxicity is thought to involve a mechanism similar to the mechanism of the organophosphates; consequently, they exhibit similar shortcomings, including animal toxicity.
A major problem in pest control results from the capability of many species to develop pesticide resistance. Resistance results from the selection of naturally-occurring mutants possessing biochemical, physiological or behavioristic factors that enable the pests to tolerate the pesticide. Species of Anopheles mosquitoes, for example, have been known to develop resistance to DDT and dieldrin. DDT substitutes, such as Malathion(trademark), propoxur and fenitrothion are available; however, the cost of these substitutes is much greater than the cost of DDT.
There is clearly a longstanding need in the art for pesticidal compounds that are pest-specific, that reduce or eliminate direct and/or indirect threats to human health posed by presently available pesticides, that are environmentally compatible in the sense that they are biodegradable, and are not toxic to non-pest organisms, and have reduced or no tendency to bioaccummulate.
Many pests, including for example blood-inbibing pests, must consume and digest a proteinaceous meal to acquire sufficient essential amino acids for growth, development and the production of mature eggs. Adult pests, such as adult mosquitoes, need these essential amino acids for the production of vitellogenins by the fat body. These vitellogenins are precursors to yolk proteins which are critical components of oogenesis. Many pests, such as house flies and mosquitoes, produce oostatic hormones that inhibit egg development by inhibiting digestion of the protein meal, and thereby limiting the availability of the essential amino acids necessary for egg development.
Serine esterases such as trypsin and trypsin-like enzymes (collectively referred to herein as xe2x80x9cTTLExe2x80x9d) are important components of the digestion of proteins by insects. In the mosquito, Aedes aegypti, an early trypsin that is found in the midgut of newly emerged females is replaced, following the blood meal, by a late trypsin. A female mosquito typically weighs about 2 mg and produces 4 to 6 xcexcg of trypsin within several hours after a ingesting blood meal. Continuous boisynthesis at this rate would exhaust the available metabolic energy of a female mosquito; as a result, the mosquito would be unable to produce mature eggs, or even to find an oviposition site. To conserve metabolic energy, the mosquito regulates TTLE biosynthesis with a peptide hormone named Trypsin Modulating Oostatic Factor (TMOF). TMOF mosquitoes produce in the follicular epithelium of the ovary 12-35 hours after a blood meal; TMOF is then released into the hemolymph where it binds to a specific receptor on the midgut epithelial cells, signaling the termination of TTLE biosynthesis.
This regulatory mechanism is not unique for mosquitoes; flesh flies, fleas, sand flies, house flies, dog flies and other insect pests which need protein as part of their diet have similar regulatory mechanisms.
In 1985, Borovsky purified an oostatic hormone 7,000-fold and disclosed that injection of a hormone preparation into the body cavity of blood imbibed mosquitoes caused inhibition of egg development and sterility (Borovsky, D. [1985] Arch. Insect Biochem. Physiol. 2:333-349). Following these observations, Borovsky (Borovsky, D. [1988] Arch. Ins. Biochem. Physiol. 7:187-210) reported that injection or passage of a peptide hormone preparation into mosquitoes inhibited the TTLE biosynthesis in the epithelial cells of the gut. This inhibition caused inefficient digestion of the blood meal and a reduction in the availability of essential amino acids translocated by the hemolymph, resulting in arrested egg development in the treated insect. Borovsky observed that this inhibition of egg development does not occur when the oostatic hormone peptides are inside the lumen of the gut or other parts of the digestive system (Borovsky, D. [1988], supra).
Following the 1985 report, the isolated hormone, (a ten amino acid peptide) and two TMOF analogues were disclosed in U.S. Pat. Nos. 5,011,909 and 5,130,253, and in a 1990 publication (Borovsky, et al. [1990] FASEB J. 4:3015-3020). Additionally, U.S. Pat. No. 5,358,934 discloses truncated forms of the full length TMOF which have prolines removed from the carboxy terminus, including the peptides YDPAP (SEQ ID NO: 1), YDPAPP (SEQ ID NO: 1), YDPAPPP (SEQ ID NO: 1), and YDPAPPPP (SEQ ID NO: 1).
Neuropeptides Y (NPY) are an abundant family of peptides that are widely distributed in the central nervous system of vertebrates. NPY peptides have also been recently isolated and identified in a cestode, a turbellarian, and in terrestrial and marine molluscs (Maule et al., 1991 xe2x80x9cNeuropeptide F: A Novel Parasitic Flatworm Regulatory Peptide from Moniezia expansa (Cestoda: Cyclophylidea)xe2x80x9d Parasitology 102:309-316; Curry et al., 1992 xe2x80x9cNeuropeptide F: Primary Structure from the Turbellarian, Arthioposthia triangulataxe2x80x9d Comp. Biochem. Physiol. 101C:269-274; Leung et al., 1992 xe2x80x9cThe Primary Structure of Neuropeptide F (NPF) from the Garden Snail, Helix aspersaxe2x80x9d Regul. Pep. 41:71-81; Rajpara et al., 1992 xe2x80x9cIdentification and Molecular Cloning of Neuropeptide Y Homolog that Produces Prolonged Inhibition in Aplysia Neuronsxe2x80x9d Neuron. 9:505-513).
Invertebrate NPYs are highly homologous to vertebrate NPYs. The major difference between vertebrate and invertebrate NPYs occurs at the C-terminus where the vertebrate NPY has an amidated tyrosine (Y) whereas invertebrates have an amidated phenylalanine (F). Because of this difference, the invertebrate peptides are referred to as NPF peptides.
Cytoimmunochemical analyses of NPY peptides suggest that they are concentrated in the brain of various insects, including the Colorado potato beetle Leptinotarsa decemlineata (Verhaert et al., 1985 xe2x80x9cDistinct Localization of FMRFamide- and Bovine Pancreatic Polypeptide-Like Material in the Brain, Retrocerebal Complex and Subesophageal Ganglion of the Cockroach Periplaneta americanaxe2x80x9d L. Brain Res. 348:331-338; Veenstra et al., 1985 xe2x80x9cImmunocytochemical Localization of Peptidergic Neurons and Neurosecretory Cells in the Neuro-Endocrine System of the Colorado Potato Beetle with Antisera to Vertebrate Regulatory Peptidesxe2x80x9d Histochemistry 82:9-18). Partial purification of NPY peptides in insects suggests that both NPY and NPF are synthesized in insects (Duve et al., 1981 xe2x80x9cIsolation and Partial Characterization of Pancreatic Polypeptide-like Material in the Brain of the Blowfly alliphora vomitoriaxe2x80x9d Biochem. J. 197, 767-770).
Researchers have recently isolated two neuropeptides with NPF-like immunoreactivity from brain extracts of the Colorado potato beetle. The researchers purified the peptides using C18 reversed phase high pressure liquid chromatography (HPLC), and determined their structure using mass spectrometry. The deduced structures of these peptides are: Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 1) and Ala-Pro-Ser-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2) designated NPF I and NPF II, respectively (Spittaels etal., 1996).
The present inventors have surprisingly discovered that NPF adversely affects TTLE biosynthesis in the midgut of female Aedes aegypti fed a blood meal and injected with NPF polypeptide. Because the structure of NPF is different from TMOF it appears that NPF does not bind to a TMOF-specific binding site on the gut receptor but to a different site on the same or different receptor. Furthermore, cytoimmunochemical analysis, by the inventors, of the mosquito gut after the blood meal, using antiserum against NPF, has surprisingly revealed that exocrine cells with NPF-like molecules that are synthesized by mosquito epithelial cells 24 hours after a blood meal. NPF therefore appears to be a secondary signal in a cascade of signals: first TMOF is released from the ovary, TMOF then binds to a TMOF gut receptor (Borovsky et al., 1994) that stimulates the synthesis and release of NPF from gut specific exocrine cells. NPF then binds to a receptor site on the gut at a site which may be adjacent to or part of the TMOF receptor, resulting cessation of biosynthesis of TTLE. This surprising discovery opens the door to a new generation of NPF pesticides, which inhibit biosynthesis of TTLE in a more direct manner than previously disclosed TMOF peptides.
The present invention provides novel compositions comprising novel pesticidal compounds. The compounds are preferably polypeptides, such as peptides or proteins. In a preferred embodiment, these pesticidal compounds inhibit digestion in pests by inhibiting synthesis of pest digestive enzymes, such as TTLE. In a specific embodiment, these compounds can be used to control populations of pests, such as populations of blood-ingesting insects.
In one aspect, the compositions of the present invention comprise a pesticidal polypeptide which comprises an amino acid sequence having a formula:
A1A2A3A4A5FLNKxe2x80x83xe2x80x83(Formula I)
wherein:
A1 is selected from the group consisting of Y, A, D, F, G, M, P, S and Y;
A2 is selected from the group consisting of A, D, E, F, G, N, P, S and Y;
A3 is optionally present and is selected from the group consisting of A, D, F, G, L, P, S and Y;
A4 is optionally present when A3 is present and is selected from the group consisting of A, F, G, L and Y;
A5 is optionally present when A4 is present and is selected from the group consisting of A, F, L and P;
FLNK is a flanking region which is optionally present and is selected from the group consisting of: P, PP, PPP, PPPP, and PPPPP;
The pesticidal polypeptide preferably does not consist of YDPAP6, DYPAP6, PAP6, YDPAP, YDPAP2, YDPAP3, YDPAP4, NPTNLH or DF-OMe.
In a narrower aspect the pesticidal polypeptide comprises an amino acid sequence which consists essentially of the amino acid sequence of Formula I. In a preferred aspect, the pesticidal polypeptide comprises a TMOF fragment TMOF amino acids adjacent to the amino acid sequence of Formula I. The fragment preferably has less than 50% of the number of amino acid residues of full-length native TMOF, preferably 2-5 amino acid residues. In still another aspect, the pesticidal polypeptide consists of the amino acid sequence of Formula I.
In another aspect, the present invention pertains to DNA sequences encoding the pesticidal polypeptides disclosed herein. Such DNA sequence can be used as known in the art to provide transformed plants or other food organisms which express a pesticidal polypeptide of the present invention.
The subject invention provides pest control compositions comprising pesticidal polypeptides formulated for application to the target pests or their situs. In a specific embodiment, prokaryotic or eukaryotic recombinant hosts which express a pesticidal polypeptide are provided by the subject invention. In a specific example, yeast or algae (preferably unicellular siliceous or green algae) are transformed to express a pesticidal polypeptide of the present invention. The transformed hosts can, for example, be applied to water areas where insect level such as mosquito larvae will ingest the transformed host, resulting in control of the mosquitoes by the pesticidal polypeptide. Furthermore, the polynucleotides of the present invention can be used to modify a virus, which may be used to deliver the polynucleotides to pest or other cells.
Another aspect of the present invention pertains to a method of controlling pests comprising administering to said pest or applying to a pest-inhabited locus an effective amount of a pesticidal polypeptide of the present invention.
The pesticidal polypeptides of the invention are also useful in controlling pest populations in areas of infestation, or areas susceptible to infestation and/or combating target pest populations, and can be employed along with pest repellents and pest attractants to control a pest population in a geographical area.
The invention also includes pesticidal compositions which contain one or more of the pesticidal polypeptides described above, including one or more of the pesticidal polypeptides and a pesticidally acceptable carrier, and also includes methods of killing or controlling insects which involve applying to the insects or their environment such pesticidal compositions. In one aspect, the pesticidal compositions of the present invention are administered in the form of a spray or a time release dosage unit. The pesticidal compositions can also comprise various other known pesticidal polypeptides or other pesticides targeting the same or different pests.
Methods of making pesticidal compositions are also included within the scope of the present invention which comprise bringing one or more of the said pesticidal polypeptides into association with a suitable carrier, diluent or excipient therefor.
As used herein, the term xe2x80x9cpesticidally effectivexe2x80x9d is used to indicate an amount or concentration of a pesticide which is sufficient to reduce the number of pests in a geographical locus, as compared to a corresponding geographical locus in the absence of the amount or concentration of the pesticide.
The term xe2x80x9cpesticidalxe2x80x9d is not intended to refer only to the ability to kill pests, but also includes the ability to interfere with a pests life cycle in any way that results in an overall reduction in the pest population. For example, the term xe2x80x9cpesticidalxe2x80x9d included inhibition or elimination of reproductive ability of a pest, as well as inhibition of a pest from progressing from one form to a more mature form, e.g., transition between various larval instars or transition from larvae to pupa or pupa to adult. Further, the term pesticidal is intended to include all phases of a pest life cycle; thus, for example, the term includes larvicidal, ovicidal, and adulticidal action.
As used herein, the term xe2x80x9cpest attractingxe2x80x9d in reference to chemical or physical attractant (e.g., light attractant) means that the density of pests in an area in the presence of the attractant is greater than the density of pests in a corresponding area without the attractant. As used herein, the term xe2x80x9cpest repellingxe2x80x9d is correspondingly intended to indicate that the density of pests in an area in the presence of the repellent is lower than the density of pests in a corresponding area in the absence of the repellent.
The word xe2x80x9ctransformxe2x80x9d is broadly used herein to refer to introduction of an exogenous polynucleotide sequence into a prokaryotic or eukaryotic cell by any means known in the art (including, for example, direct transmission of a polynucleotide sequence from a cell or virus particle as well as transmission by infective virus particles and transmission by any other known means for introducing a polynucleotide into a cell), resulting in a permanent or temporary alteration of genotype and in an immortal or non-immortal cell line.
The terms xe2x80x9cpeptide,xe2x80x9d xe2x80x9cpolypeptide,xe2x80x9d and xe2x80x9cproteinxe2x80x9d as used herein are intended to refer to amino acid sequences of any length.
The term xe2x80x9cpesticidal polypeptidexe2x80x9d is used herein to indicate polypeptides comprising NPF and TMOF peptides, as well as fragments, derivatives and analogues and other functional equivalents of NPF and TMOF.
The methods and materials of the present invention provide a novel approach to controlling insects and insect-transmitted diseases. The peptides of the present invention have advantageous activity over previously disclosed compounds.